HU214742B - Process for producing immunogenic peptides and pharmaceutical compositions comprising same - Google Patents

Abstract

This invention relates to a peptide suitable for vaccination of mammals, such as pigs, against the hormone LHRH and comprising at least two LHRH sequences in tandem.

Description

The present invention relates to the production of a peptide which is capable of releasing a serum component (LHRH = luteinising hormone releasing hormone, also known as GnRH = gonadotropin releasing hormone) effective against enterinizing hormone releasing hormone (LHRH).

The present invention also relates to a composition and vaccine or medical preparation (vaccines and pharmaceuticals) based on such a peptide and to the immunization of mammals against LHRH and the improvement of pork quality using these vaccines and medical preparations.

The LHRH hormone is a small peptide, a chain of 10 amino acids (i.e., a decapeptide) and has the following amino acid sequence (usually the amino terminus to the left and the carboxy terminus to the right): pGlu-His-TrpSer-Tyr- Gly-Leu-Arg-Pro-Gly-NH ₂

Above, the amino acids are indicated by the three-letter code, or can also be the one-letter code where #E is pyroglutamic acid and G 9 is glycinamide.

# EHWSYGLRPG0

It is known that LHRH, when combined with a carrier protein, can be used to vaccinate mammals. Such inoculations may be performed for a variety of purposes, all of which are related to the natural function of LHRH. LHRH produced in the hypothalamus regulates the production of sex hormones LH (luteinising hormone) and FSH (follicle stimulating hormone) in the pituitary gland. It is known that the drastic decrease in LH and FSH blood levels in male animals inhibits the production of androgens and spermiogenesis in the testes, in females inhibits the production of progestogens and estrogens and follicular maturation in the ovaries.

Reduction of blood levels of androgens, progestogens and estrogens to levels comparable to those obtained by castration or ovariectomy can be achieved by effective immunization of the animal against LHRH.

In the veterinary field, 100% effective immunization could be used to sterilize, for example, small pets such as male and female cats, or to treat aggression in male dogs instead of drastic surgery such as castration or ovariectomy. Another conceivable area of application for immunization against LHRH in dogs is to prevent firing and, in bullfighting, to prevent anxiety. In human medicine, immunization against LHRH can be used to treat prostate and breast cancer and, if necessary, to treat some forms of pituitary carcinoma.

In addition, anti-LHRH serum can be used in animal husbandry, namely for fattening pigs for slaughter. The meat of male, sexually mature pigs has a characteristic smell. The sexually mature pig's testicles produce many steroids which are stored in adipose tissue. These steroids (Cl9 16 steroids) are responsible for the unwanted, urine-like odor that is released when roasting meat [Brooks et al., J. Amin. Sci. 62, 1279 (1986)]. Due to this undesirable "zing", the meat of sexually mature male pigs is barely, if at all, fit for consumption. As about 10% of pigs are sexually mature before slaughter, this option causes a great deal of loss for pig farmers. To prevent this loss, all tapings are castrated at an early age - without anesthesia.

Apart from the fact that such castration is an unpleasant procedure for animals, castration also causes growth inhibition and the quality of the meat is lower than that of intact animals (until the intact animal has reached sexual maturity).

An animal-friendly alternative (which additionally improves the quality of the meat) is the immunization of young animals against LHRH, which results in a decrease in the concentration of LHRH in the young animal's pituitary gland.

This decrease in LHRH concentration in the pituitary gland leads to a decrease in the blood levels of biologically active LH and FSH, which results in the growth animal being prevented or delayed from developing testicles and producing less steroids. Vaccination with effective LHRH therefore avoids the development of unwanted cochlea in pigs before the time of slaughter.

Recently, it is becoming common practice in the male animals to delay or stop testicular development by inoculating a carrier protein-bound LHRH. However, results are variable when using known serum formulations, such as those based on LHRH itself or an analogue such as [Dtrp ⁶ ] LHRH. (Chaffaux et al., Recueil de Medecine Vétérinaire 16, 133-145 (1985)). For example, there are vaccinated animals that have little or no response to serum. When used in young livers, good serum is required to prevent the development of litter in young litters so that no litter develops (even for 35 weeks after birth), even in a very large pig population. Known serum formulations do not meet this requirement.

This also applies to the immunogenic LHRH serum described in U.S. Patent No. 4,608,251. The serum recommended there is based on a nonav or decapeptide having the structural formula: (C) KW SYGLRPG6, or on the dimer of decapeptides, which may be formed by the coupling of amino-terminated cysteines and corresponds to the following structural formula: CKWSYGLRPG3

CKWSYGLRPG0

This dimer does not appear to be more effective than the monomer peptides.

International Patent Application Publication No. WO 88/05308 proposes LHRH sera based on LHRH partial peptides of 5, 6 or 7 amino acids in length, particularly peptides that contain both the amino-terminated pGlu and the carboxy-terminated Gly-NH. _{2 are} included. An example of such sub-peptides (given by one letter code):

GB 214 742 Β #EH W S Y, #EHWSYGL, #EHWSYGL, HWSYGLR, WSYGLR, SY GLRPGS, and Y G L RP G 9. However, sera based on these sub-peptides also exhibit the above disadvantages.

Surprisingly, it has been found that a better and mainly more reliable serum is obtained if the double LHRH is based on a peptide having at least two successive LHRH sequences.

The present invention thus relates to a process for pGlu-His-Trp'-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gln-His-Trp ^2- Ser-Tyr-Gly-Leu-Arg-Pro-GlyCys-NH ₂ or Peptides of Formula pGlu-His-Trp'-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gln-His-Trp- ^2- Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH ₂ - Trp ¹ and Trp ² are for the production of Trp or N-formyl-Trp, characterized in that the corresponding protected amino acids, or sequences formed therefrom, are linked in the desired order in a known manner and then deprotected.

The invention also relates to the preparation of a composition comprising the peptide of the invention in an immunized version. There are various methods known to those skilled in the art for rendering non-immunogenic substances per se immunogenic. In a preferred embodiment, the peptide of the invention is coupled to a suitable carrier protein. The C or N terminus may be suitably used for chemical bonding. Those skilled in the art will know exactly which coupling method and carrier proteins are appropriate. See, for example, U.S. Patent 4608251, cited above, and WO 88/05308. Preferred compounds of the present invention are those comprising an immunogenic conjugate of a protein and a peptide of the invention. Other options for altering the peptide are by cross-linking to a larger complex or by making the peptide as part of a larger protein using a recombinant DNA molecule. The invention thus also relates to a chemical ethylene containing an inununogenic complex and an immunogenic recombinant protein to which a peptide of the invention belongs.

Of course, the invention also relates to sera and pharmaceutical compositions which comprise a compound of the present invention in combination with at least one immunoadjuvant. Suitable adjuvants are known to those skilled in the art and include, for example, Complete Freund's Adjuvants (CFA) or Incomplete Freund's Adjuvants (IFA).

The compounds of the present invention, or compositions containing them, can be used to immunize mammals against LHRH, which method comprises inoculating the mammals with the serum or pharmaceutical composition of the invention. The indications for this vaccine have already been discussed above, such as its use in human medicine for the treatment of certain forms of prostate and breast carcinoma and pituitary carcinoma, its various uses in veterinary medicine and its various uses in animal husbandry. A particular area of its use is to improve the quality of pork, which is characterized in that the pigs are inoculated with the serum preparation of the invention.

The invention is further illustrated by the following examples.

Example 1 pGlu-His-Trp'-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gln-His-Trp ^2- Ser-Tyr-Gly-Leu-Arg-Pro-Gly-CysNH ₂

The solvents and reagents used were of analytical purity and were purchased from Merck (Darmstadt), Germany, which were not subjected to further purification. The dichloromethane (DCM) was purified on a column packed with activated basic alumina. Boc-protected amino acids, 4-methylbenzhydrylamine (MBHA) resin, and benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate (BOP, Castro's reagent, (Le-Nguyen D. , Heitz A. and Castro B. (1987) Renin Substrates, Volume 2, Rapid Solid Phase Synthesis of a Tetradecapeptide Using a BOP Reagent, J. Chem. Soc. Perkin Trans I, 1915-1919; Fournier A. , Wang C. T. and Felix AM (1988) Applications of BOP Reagent in Solid Phase Synthesis, Int. J. Peptide Protein Res. 31, 86-97.) From Novabiochem.

The synthesis was carried out in a 250 ml glass vessel with a glass filter at the bottom. The washes, BOC deprotections and couplings were performed manually. The coupling was performed with BOP reagent and in situ neutralization using diisopropylethylamine (DIEA) (here call 1).

During the preparation, MBHA resin (1% divinylbenzene, 0.75 meq / mg, 5.1 g) was swollen in 50% DMF / DCM for 24 hours and washed with DCM.

Subsequently, the following reagents were coupled sequentially using the following coupling sequence: Boc-Cys (Mob), Boc-Gly, Boc-Pro, Boc-Arg (Tos), Boc-Leu, Boc-Gly, Boc-Tyr ( 2-BrZ), Boc-Ser (Bzl), Boc-Trp (For), BocHis (Tos), Boc-Gln, Boc-Gly, Boc-Pro, Boc-Arg (Tos), Boc-Leu, Boc-Gly , Boc-Tyr (2-BrZ), Boc-Ser (Bzl), Boc-Trp (For), Boc-His (Tos) and Boc-pGlu.

Switching sequence:

1. DCM Wash (2 minutes twice).

2. Wash with 50% (v / v) TFA / DCM (once for 2 minutes).

3. Nitrogen bubbling (50 minutes once) in 50% TFA / DCM.

4. DCM wash (twice 2 min).

5. Wash with isopropanol (2 minutes twice).

6. Dimethylformamide wash (2 x 2 min).

7. Coupling step with nitrogen bubbling (90 minutes).

8. DCM Wash (2 min twice).

The amino acid analysis of the resulting peptide is consistent with the composition resulting from the administration.

HU 214 742 Β

Example 2 pGlu-H is-Trp ¹ -Ser-Tγ-Gly-Leu-Arg-Pro-Gly-Gln-His-Trp ² -Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH ₂

Example 1 was performed with the difference that Boc-Cys (Mob) reagent was omitted, i.e., Boc-Gly reagent was used for coupling 1.

The amino acid analysis of the resulting peptide is consistent with the composition resulting from the administration.

Functional example

Three groups of male newborn pigs (5 animals per group) were vaccinated with LHRH serum after birth (day 0). The sera consisted of a peptide linked via a C-terminal cysteine and a carrier protein, KLH (Keyhole Limpet Hemocyanine). This coupling was effected by MBS (m-malcimidobenzoyl-N-hydroxysuccinimide ester, 1. Geysen et al., PNAS 81, 3998-4002 (1984)).

Proteins used:

Group I: LHRH Formula #EHWSYGLRPGCS

II. group: [D-Trp ⁶ ] LHRH formula: #EHWSY [D-Trp] LRPG Cd

III. group: two LHRHs in a row, formula:

ffiHWSYGLRPGQHWSYGLR PGC 9

After binding to KLH (12 mg peptide to 1 mg KLH), the peptides were emulsified in 1 ml CFA (1 ml peptide-KLH solution in 1 ml CFA). The emulsion was administered intramuscularly on day 0 and 8 weeks later. At the second inoculation, IFA was used.

One control group (Group IV) was inoculated with vehicle protein (KLH) and adjuvant alone.

On day 0, and after 4, 8 and 12 weeks, the size of the testicles was measured. 17-18. at week 7 the animals were slaughtered. At 12 weeks after the first inoculation, testosterone levels were measured in pig serum. At day 131 after the first inoculation, weights of testes and parotid and vesicula seminalis (seminal vesicle) and bulbourethral gland were measured in the animals.

The results of the four groups were as follows: After inoculation of LHRH (group I), the external examination showed that three of the 5 animals had smaller testicles than the control group (group IV). In two of the three small testicular animals, the testicles became so small that they could no longer be measured by external examination.

Of the [D-Trp ⁶ ] LHRH-vaccinated animals (Group II), 2 of the 5 animals had apparently smaller testicles than control animals, and in one animal the testicles were no longer measurable.

Each of the 5 animals vaccinated with two related LHRHs had a smaller testicular size compared to control animals, and testicles were no longer measurable in 4 animals.

The average testosterone levels in the serum are:

2.47 (1.21) pmol / ml (control group, 5 animals)

1.89 (2.24) pmol / ml (Group I, 5 animals)

2.72 (2.23) pmol / ml (Group II, 5 animals)

0.51 (0.08) pmol / ml (Group III, 5 animals).

Mean testicular and epididymis masses: 258 (50) g / 55 (11) g (control group, 5 animals)

110 (123) g / 21 (17) g (Group I, 5 animals)

221 (88) g / 42 (12) g (Group II, 5 animals) (11) g / 11 (2) g (Group III, 4 animals).

Masses of mean seminal vesicle and bulbourethral gland, respectively:

135 (44) g / 119 (24) g (control group, 5 animals) (80) g / 37 (48) g (group I, 5 animals)

137 (71) g / 135 (52) g (Group II, 4 animals) (3) g / 15 (5) g (Group III, 4 animals).

Values in parentheses are standard deviations.

Since delayed testicular development or even regrowth of testes may be directly related to a decrease in the pig's androgen production capacity and consequently in the development of undesirable cochlea, the desired complete protection is provided in Annex III. group, where the dual LHRH serum of the invention was used, while only partial protection was observed in Groups I and II. group, which were used for comparison.

Claims (3)

PATENT CLAIMS 1. Procedure pGlu-His-Trp'-Ser-Tyr-Gly-Leu-ArgPro-Gly-Gln-His-Trp ^2- Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Cys -NH ₂ or pGlu-His-Trp'-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Gln-His-Trp ² -Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH ₂ peptides, wherein Trp ¹ and Trp ² are Trp or N-formyl-Trp, characterized in that the corresponding protected amino acids or sequences formed therefrom are linked in a known manner in the desired order and then deprotected. A process for the preparation of a pharmaceutical composition comprising the step of forming a peptide or immunogenic form thereof according to claim 1 in admixture with a carrier and / or excipient conventionally used in the preparation of a pharmaceutical composition to form a pharmaceutical composition. 3. The method of claim 2, wherein the pharmaceutical composition is for immunocastration of a male mammal.